Calmodulin (CaM) is a multifunctional calcium receptor in all eukaryotic cells and is involved in fundamental biological processes as diverse as neurotransmission, muscle contraction, and cell division. Elucidating the means by which the concentration and activity of CaM are regulated would, therefore, greatly aid the understanding of a variety of basic biological events. Our specific interest is in the role CaM plays in neuronal differentiation. In the present studies, the role of CaM is examined by studying the effects of differentiating agents on the synthesis and intracellular distribution of CaM and the effects of altering CaM levels on neuronal growth and differentiation. Prominent among the factors that regulate the synthesis of CaM are the multiple genes that express CaM. Three distinct CaM genes, expressing five different CaM mRNAs, have been identified in mammalian cells. Accordingly, the factors controlling the expression and distribution of these CaM mRNAs and the functional consequences of altering the CaM mRNAs in cells will be examined. Specifically, using the pheochromocytoma PC12 cell line as a model, the role CaM plays in nerve growth factor (NGF)- induced differentiation will be studied by determining the effects of NGF on the amount and localization of CaM and the different CaM mRNAs. These parameters will be compared with those induced by cyclic AMP, which causes morphological changes in PC12 cells similar to but not identical to those caused by NGF. The levels of the individual CaM mRNAs will be altered by introducing oligodeoxynucleotide sequences which are antisense to the different CaM mRNAs in PC12 cells and by transfecting cells with expression vectors containing the cDNA sequences for the different CaM mRNAs. By selectively altering the activity of the different CaM mRNAs and by measuring the functional consequences of these alterations, such as changes in the intracellular distribution of CaM, it should be possible to determine whether the different CaM transcripts play different functional roles. The biological activity, amount, and subcellular localization of CaM will be determined by bioassay, radioimmunoassay and immunocytochemical techniques, respectively. The CaM mRNAs will be detected using synthetic radiolabeled oligonucleotide probes directed at specific sites on each of the CaM mRNAs. The amount and localization of CaM mRNAs will be assessed with Northern analysis and in situ hybridization techniques. The functional consequences of altering the CaM mRNAs in PC12 cells will be assessed by measuring certain morphological and biochemical sequelae of neuronal differentiation. These include changes in cellular proliferation and differentiation, and changes in the activity of certain CaM-associated proteins (the CaM-binding protein GAP-43 and CaM-dependent protein kinase II) thought to be involved in cellular differentiation. Finally, studies using sympathetic and hippocampal cells in culture are planned in order to determine whether the general principles of CaM gene regulation uncovered using the PC12 cells also apply to primary neuronal cells. These studies will not only provide additional information on the role CaM and its mRNAs play in cellular growth and differentiation, but also, through the use of antisense oligonucleotides to inhibit the different CaM mRNAs, may suggest a novel means by which the intracellular concentration of CaM can be selectively controlled with pharmacological agents.